Varicella-zoster virus (VZV) is an ancient virus. Estimations of its origins have established that the modem herpesviruses arose some 60-80 million years ago. VZV is a member of the alphaherpesvirus subfamily of herpesviridae. It is the etiologic agent of chickenpox in childhood, after which the virus enters a latent state in the dorsal root ganglia; decades later, the same virus reactivates and causes the disease shingles (herpes zoster). The entire sequence of the 125 kbp VZV genome has been published (see Davison et al., J. Gen. Virol., 67:1759-1816 (1986)). With the subsequent publication of sequence data from other herpesviruses, the alphaherpesvimuses have now been subdivided into two genera called Simplexvirus and Varicellovirus. VZV is considered to have one of the most stable genomes of all herpesviruses. The Oka strain of varicella vaccine derived from a Japanese child with chickenpox has a few minor genomic differences from North American strains, but to date no antigenic variation has been discovered amongst the major surface immunogens of the virion (Arvin et al., Annu. Rev. Microbiol., 50:59-100 (1996)).
Based on their extensive analyses of herpesviral molecular evolutionary history, it has been estimated that herpesvirus DNA sequences mutate 10-100 times faster than the equivalent classes of sequences on the host genome. For glycoprotein gB, a highly conserved open reading frame (ORF) among all herpesviruses, it has been calculated that nonsynonymous substitutions have occurred at a rate of 2.7xc3x9710xe2x88x928 substitutions per site per year and synonymous substitutions at 10xe2x88x927 substitutions per site per year. Convincing arguments have been made in favor of the concept of cospeciation; in other words, herpesvirus lineages arise by way of co-evolution with their specific host. In the case of VZV, the progenitor virus most likely arose 60-70 million years before the present.
Of all the human herpesviruses, VZV may undergo the fewest replication cycles during the lifetime of the infected host. Based on a probable schema of pathogenesis, the virus actively replicates for a period of 10-14 days after infection of the human host. During a bout of chickenpox, therefore, VZV has at most 20 replication cycles. Based on the current understanding of VZV latency and reactivation, no further replication occurs unless the individual develops herpes zoster in late adulthood. Because of the above scenario, the genetic stability of the VZV genome has been presumed.
VZV contains the smallest genome of the human herpesviruses, containing about 70 ORFs within the complete VZV-Dumas sequence. Of these ORFs, at least seven code for glycoproteins, of which glycoprotein B (gB), glycoprotein E (gE), glycoprotein H (gH), and glycoprotein I (gI) are present on the exterior of the virion. VZV gE, in complex with glycoprotein I (gI), acts as a human Fc receptor on the surface of infected cells (Litwin et al., J. Virol., 66:3643-51 (1992), Litwin et al., Virology, 178:263-72 (1990)). The cytoplasmic tails of both gE and gI contain endocytosis motifs, allowing internalization and recycling of the complex to and from the cell (Olson et al., J. Virol., 71:110-119 (1997), Olson et al., J. Virol., 71:4042-4054 (1992)). The gE and gI cytoplasmic tails also are modified by both serine/threonine and tyrosine phosphorylation motifs. The fact that gE cannot be deleted suggests that it is essential (Cohen et al., Proc. Natl. Acad. Sci. USA, 90:7376-7380 (1993), Mallory et al., J. Virol., 71:8279-88 (1997)).
In VZV infection in humans, VZV gE is the most abundantly produced viral glycoprotein during infection. VZV gE is a major antigenic determinant to which numerous humoral and cytolytic responses are observed (Arvin et al., J. Immunol., 137:1346-1351 (1986); Bergen et al., Viral Immunol., 4:151-166 (1991); and Ito et al., J. Virol., 54:98-103 (1985)). Recently, an immunodominant B-cell epitope was demarcated in the gE ectodomain; the epitope is defined by murine monoclonal antibody (MAb) 3B3 (Duus et al., J. Virol., 70:8961-8971 (1996); Hatfield et al., BioTechniques 22:332-337 (1997); and Grose, U.S. Pat. No. 5,710,348).
It has long been believed that varicella zoster virus exists in nature as a single serotype (Rentier, Neurol., 45(Suppl. 8), S8 (1995), and that all varicella zoster viruses had essentially the same immunological properties. The first strain of varicella zoster virus that was sequenced was VZV-Dumas. Following the publication of this sequence, it was further believed that all varicella zoster viruses had essentially the same genetic properties as VZV-Dumas.
Significant progress has been made in the diagnosis of and vaccination against the sole VZV serotype that is believed to exist and cause disease in the United States. However, the production of the reagents used in diagnosis and vaccination of VZV is time consuming and expensive due to the slow growth rate of the strain grown to produce antigens for diagnostic and vaccine use.
The present invention represents a significant advance in the art of detecting and preventing varicella zoster virus infection and disease. During the characterization of a varicella zoster virus isolated from a patient, the surprising and unexpected observation was made that the virus had a different serotype. This strain was designated VZV-MSP. The molecular basis of the different serotype was found to be a single nucleotide polymorphism in the genome between VZV-Dumas and VZV-MSP. It was also determined that this single nucleotide polymorphism resulted in the loss of an epitope that is the epitope to which most protective antibody is produced upon vaccination with most currently used vaccines.
Typically, varicella zoster virus isolates can be divided into two groups with respect to growth rate in tissue culture cells. Some isolates, for instance VZV-Oka and VZV-Ellen, grow at a rate that results in complete lysis of a monolayer in about 5 to 7 days. Clinical isolates typically grow at a rate that results in complete lysis of a monolayer in about 4 to 5 days. Further investigation revealed that the new strain, VZV-MSP, unexpectedly and surprisingly had by in vitro tissue culture a growth rate that was significantly higher than previously characterized isolates, and was able to lyse a monolayer in about 2 days.
The present invention provides a method for detecting antibodies that specifically bind to a varicella zoster polypeptide. A biological sample that includes an antibody is contacted with a preparation that includes a varicella zoster polypeptide, for instance an isolated varicella zoster polypeptide or fragment thereof, to form a mixture. The varicella zoster polypeptide includes a polymorphism and can encoded by a polymorphism of ORF37. The polymorphism in the polypeptide encoded by the polymorphic ORF37 can be due to a single amino acid polymorphism, which can be present in the polypeptide as a leucine at amino acid 269. Alternatively, the varicella zoster polypeptide includes a polymorphism and can encoded by a polymorphism of ORF68. The polymorphism in the polypeptide encoded by the polymorphic ORF68 can be due to a single amino acid polymorphism, which can be present in the polypeptide as an asparagine at amino acid 150. The mixture is incubated under conditions to allow the antibody to specifically bind the polypeptide to form a polypeptide:antibody complex. The presence or absence of the polypeptide:antibody complex is then detected. Detecting the polypeptide:antibody complex indicates the presence of antibodies that specifically bind to a varicella zoster polypeptide.
The preparation can include whole varicella zoster virus, for instance VZV-MSP or a modified varicella zoster virus, where the modified virus has the ATCC designation VR-795 wherein the nucleotide sequence of the virus has been modified to comprise the polymorphism of ORF37 or ORF68. The biological sample can be blood, vesicle fluid, bone marrow, brain tissue, or combinations thereof. Also provided are kits for detecting antibodies that specifically bind to a varicella zoster polypeptide. This kits include a whole varicella zoster virus.
In another aspect, the present invention provides a method for detecting the presence of a varicella zoster virus in an animal. The method includes detecting the presence of an antibody to a varicella zoster virus polypeptide encoded by a polymorphic ORF of GenBank X04370. The ORF can be ORF37 or ORF68, where the encoded polypeptide includes a single amino acid polymorphism. When the polypeptide is encoded by ORF37, the single amino acid polymorphism present in the polypeptide can be a leucine at amino acid 269. When the polypeptide is encoded by ORF68, the single amino acid polymorphism present in the polypeptide can be an asparagine at amino acid 150. Optionally, the antibody that is detected does not specifically bind to the varicella zoster polypeptide encoded by ORF37 of GenBank Accession X04370 or ORF68 of GenBank Accession X04370.
The present invention is also directed to a method for diagnosing a disease, for instance chicken pox and shingles, caused by varicella zoster virus. The method includes contacting a polynucleotide, optionally an isolated polynucleotide, of a subject suspected of having a disease caused by varicella zoster virus with a primer pair. This is incubated under conditions suitable to form a detectable amplification product, and the primer pair will not form a detectable amplification product when incubated with a polynucleotide having the nucleotide sequence of GenBank Accession X04370. An amplification product is detected, where the detection indicates that the subject has a disease caused by varicella zoster virus. The polynucleotide of the subject can be present in a biological sample, including blood, vesicle fluid, bone marrow, brain tissue, or combinations thereof.
The polynucleotide that is amplified to result in a detectable amplification product can include a single nucleotide polymorphism relative to the nucleotide sequence of GenBank Accession X04370 (SEQ ID NO:76). The primer pair can include a first primer that includes nucleotides that hybridize with a polynucleotide of GenBank Accession X04370, and a second primer comprising nucleotides that hybridize with a polynucleotide of GenBank Accession X04370, with the proviso that the 3xe2x80x2 nucleotide of the second primer hybridizes to the single nucleotide polymorphism relative to the nucleotide sequence of GenBank Accession X04370 and does not hybridize with the corresponding nucleotide present in the nucleotide sequence of GenBank Accession X04370. The single nucleotide polymorphism can be present in ORF37, and the single nucleotide polymorphism can be present at nucleotide 806 of ORF37. The nucleotide at nucleotide 806 can be a thymine. The single nucleotide polymorphism can be present in ORF68, and the single nucleotide polymorphism can be present at nucleotide 448 of ORF68. The nucleotide at nucleotide 448 can be an adenine. An example of a primer pair is CGATGACAGACATAAAATTGTAAATGTGA (SEQ ID NO:1) and CACCCAAGTATTGTTTTTCTGTCCG (SEQ ID NO:2). nucleotide of the second primer hybridizes to the single nucleotide polymorphism relative to the nucleotide sequence of GenBank Accession X04370 and does not hybridize with the corresponding nucleotide present in the nucleotide sequence of GenBank Accession X04370. The single nucleotide polymorphism can be present in ORF37, and the single nucleotide polymorphism can be present at nucleotide 806 of ORF37. The nucleotide at nucleotide 806 can be a thymine. The single nucleotide polymorphism can be present in ORF68, and the single nucleotide polymorphism can present at nucleotide 448 of ORF68. The nucleotide at nucleotide 448 can be an adenine. An example of a primer pair is
CGATGACAGACATAAAATTGTAAATGTGA (SEQ ID NO:1) and
CACCCAAGTATTGTTTTTCTGTCCG (SEQ ID NO:2).
The present invention further provides a method for detecting a varicella zoster virus, for instance VZV-MSP, having a single nucleotide polymorphism in ORF68. The method includes contacting a polynucleotide with a primer pair and incubating under conditions suitable to form a detectable amplification product. The primer pair amplifies a portion of ORF68 of GenBank Accession X04370 and/or a polymorphism thereof, that includes nucleotide 448 of ORF68. The amplification product is exposed to a restriction endonuclease having nucleotide 448 in its recognition sequence. Examples of restriction endonuclease include AflI, Asul, AvaII, Cfrl3I, Eco47I, NspIV, PshAI, Sau96I, and SinI. The amplification product is then detected. The presence of an amplification product that is not cleaved by the restriction endonuclease indicates the presence of a varicella zoster virus having a single nucleotide polymorphism in ORF68. The polynucleotide can be present in a biological sample, including, for instance, blood, vesicle fluid, bone marrow, brain tissue, or combinations thereof. Optionally, the polynucleotide can be isolated. An example of a primer pair is
GGCATACTACCAATGACACG (SEQ ID NO:12) and AAGCTCCAAGTCTCGGTGTACC (SEQ ID NO:71).
The present invention is directed to a vaccine composition that includes a modified attenuated varicella zoster virus. The modified attenuated virus has the ATCC designation VR-795, and the nucleotide sequence of the virus has been modified to contain a single nucleotide polymorphism. The single nucleotide polymorphism can be present in the coding sequence encoding glycoprotein H. For instance, the single nucleotide polymorphism in the virus can be present at nucleotide 806 of the coding sequence encoding glycoprotein H. The nucleotide present at nucleotide 806 can be a thymine. The single nucleotide polymorphism can be present in the coding sequence encoding glycoprotein E. For instance, the single nucleotide polymorphism in the virus is present at nucleotide 448 of the coding sequence encoding glycoprotein E. The nucleotide present at nucleotide 448 can be an adenine.
Also provided by the present invention is a method for producing a modified attenuated varicella zoster virus. The method includes growing the virus in a tissue culture preparation. The virus has the ATCC designation VR-795, and the nucleotide sequence of the virus has been modified to contain a single nucleotide polymorphism. The single nucleotide polymorphism can be present in the coding sequence encoding glycoprotein H. For instance, the single nucleotide polymorphism in the virus can be present at nucleotide 806 of the coding sequence encoding glycoprotein H. The nucleotide present at nucleotide 806 can be a thymine. The single nucleotide polymorphism can be present in the coding sequence encoding glycoprotein E. The single nucleotide polymorphism in the virus can be present at nucleotide 448 of the coding sequence encoding glycoprotein E. The nucleotide present at nucleotide 448 can be an adenine. The modified attenuated virus can have an in vitro growth rate that is greater than the in vitro growth rate of a second varicella zoster virus. The second varicella zoster virus can be, for instance, VZV-32, ATCC VR-586, ATCC VR-1367, or ATCC VR-795. The growth rate of the modified varicella virus can be at least about 4-fold greater than the second varicella zoster virus at 48 hours post infection. Optionally, the modified varicella virus can be isolated.
The present invention further provides isolated polynucleotides, including an isolated polynucleotide having the nucleotide sequence of nucleotides 66,074 to 68,599 of GenBank Accession X04370, with the proviso that nucleotide 66,879 is a thymine; and an isolated polynucleotide having the nucleotide sequence of nucleotides 115,808 to 117,679 of GenBank Accession X04370, with the proviso that nucleotide 116,255 is an adenine. Also provided are the isolated polypeptides encoded by each of the above two polynucleotides. The polynucleotide can be isolated from a varicella zoster virus.
Also provided are viruses having the designation VZV-MSP, VZV-VSD, VZV-VIA, or VZV-Iceland.
Definitions
As used herein, an antibody that can xe2x80x9cspecifically bindxe2x80x9d a polypeptide is an antibody that interacts only with the epitope of the antigen that induced the synthesis of the antibody, or interacts with a structurally related epitope. xe2x80x9cEpitopexe2x80x9d refers to the site on an antigen to which specific B cells and/or T cells respond so that antibody is produced. As used herein, the term xe2x80x9cpolypeptide:antibody complexxe2x80x9d refers to the complex that results when an antibody specifically binds to a polypeptide.
xe2x80x9cPolypeptidexe2x80x9d as used herein refers to a polymer of amino acids and does not refer to a specific length of a polymer of amino acids. Thus, for example, the terms peptide, oligopeptide, protein, and enzyme are included within the definition of polypeptide. This term also includes post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like. Coding sequence, coding region, and open reading frame are used interchangeably and refer to a polynucleotide that encodes a polypeptide, usually via mRNA, when placed under the control of appropriate regulatory sequences. The boundaries of the coding region are generally determined by a translation start codon at its 5xe2x80x2 end and a translation stop codon at its 3xe2x80x2 end.
An xe2x80x9cORFxe2x80x9d followed immediately by a number, for instance ORF37 or ORF68, refers to a specific open reading frame of varicella zoster virus. The approximately 70 individual open reading frames of varicella zoster virus are known to the art, and are described in Davison et al. (J. Gen. Virol., 67:1759-1816 (1986)) and at GenBank Accession X04370. GenBank Accession X04370 is also referred to herein as SEQ ID NO:76. For instance, ORF37 is the open reading frame encoded by nucleotides 66,074 to 68,599 of the nucleotide sequence at GenBank Accession X04370, and ORF68 is the open reading frame encoded by nucleotides 115,808 to 117,679 of the nucleotide sequence at GenBank Accession X04370. A xe2x80x9cpolymorphic ORFxe2x80x9d followed immediately by a number, for instance polymorphic ORF37 or polymorphic ORF68, refers to an open reading frame of varicella zoster virus that has a nucleotide sequence similar to the appropriate nucleotide sequence of GenBank X04370, but includes a single nucleotide polymorphism. Moreover, a polymorphic ORF may contain an insertion or deletion of nucleotides, preferably an insertion of 3 nucleotides or a deletion of 3 nucleotides. When referring to a specific nucleotide of an ORF, the first nucleotide of the start codon is considered to be nucleotide 1, with the following amino acids labeled consecutively. When referring herein to a specific amino acid of a polypeptide encoded by an ORF, the first methionine (prior to any post-translational modification that may occur) is considered to be amino acid 1, with the following amino acids labeled consecutively.
As used herein, the term xe2x80x9cpolynucleotidexe2x80x9d refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxynucleotides, and includes both double- and single-stranded DNA and RNA. A polynucleotide may include nucleotide sequences having different functions, including for instance coding sequences, and non-coding sequences. A polynucleotide can be obtained directly from a natural source, for instance from a virus, or can be prepared with the aid of recombinant, enzymatic, or chemical techniques. A polynucleotide can be linear or circular in topology. A polynucleotide can be, for example, a portion of a vector, such as an expression or cloning vector, or a fragment.
An xe2x80x9cisolatedxe2x80x9d polypeptide or polynucleotide means a polypeptide or polynucleotide that has been either removed from its natural environment, produced using recombinant techniques, or chemically or enzymatically synthesized. Preferably, a polypeptide or polynucleotide of this invention is purified, i.e., essentially free from any other polypeptide or polynucleotide and associated cellular products or other impurities. An xe2x80x9cisolatedxe2x80x9d varicella zoster virus means a varicella zoster virus has been removed from its natural environment, e.g, the cell that produced the virus.
As used herein, the term xe2x80x9cwhole varicella zoster virusxe2x80x9d refers to a varicella zoster virus particle or virion. The particle can be infective, i.e., be able to reproduce when introduced to an appropriate tissue culture cell under the appropriate conditions, or the particle can be inactive, i.e., incapable of reproducing.
As used herein, a xe2x80x9cbiological samplexe2x80x9d refers to a sample of tissue or fluid isolated from a subject, including but not limited to, for example, blood, plasma, serum, lymph tissue and lymph fluid, cerebrospinal fluid, bone marrow, brain tissue, samples of the skin, external secretions of the skin including vesicle fluid from a pox, organs, biopsies and also samples of in vitro cell culture constituents including but not limited to conditioned media resulting from the growth of cells and tissues in culture medium, and cell components, or combinations thereof. A xe2x80x9csubjectxe2x80x9d is an animal, including, for instance, a mouse or a human, preferably a human.
As used herein, the term xe2x80x9cwhole varicella zoster virus particlexe2x80x9d refers to an intact varicella zoster virus, for instance a varicella zoster virus that has been produced by a cell and not manipulated to cause the polypeptides that make up the envelop to disassociate from one another.
As used herein, a xe2x80x9cprimer pairxe2x80x9d refers to two single stranded polynucleotides that can be used together to amplify a region of a polynucleotide, preferably by a polymerase chain reaction (PCR). The polynucleotide that results from amplifying a region of a polynucleotide is referred to as an xe2x80x9camplification product.xe2x80x9d The phrase xe2x80x9cunder conditions suitable to form a detectable amplification productxe2x80x9d refers to the reactions conditions that result in an amplification product. For instance, in the case of a PCR, the conditions suitable to form a detectable amplification product include the appropriate temperatures, ions, and enzyme.
As used herein, the term xe2x80x9chybridizexe2x80x9d refers to the ability of two complementary single stranded polynucleotides to base pair with each other, where an adenine of one polynucleotide will base pair to a thymine of a second polynucleotide and a cytosine of one polynucleotide will base pair to a guanine of a second polynucleotide. When the term xe2x80x9chybridizexe2x80x9d is used to describe the interaction between a primer and a polynucleotide, hybridization requires that the 3xe2x80x2 nucleotide of a primer be able to base pair with the corresponding nucleotide of the polynucleotide that is to be amplified. Typically, the inability of the 3xe2x80x2 nucleotide of a primer to base pair with the polynucleotide that is to be amplified results in no amplification (see Newton et al., U.S. Pat. No. 5,595,890).
As used herein, the term xe2x80x9cin vitro growth ratexe2x80x9d refers to the rate at which a varicella zoster virus spreads from an infected tissue culture cell to an adjacent uninfected tissue culture cell. A tissue culture cell is a cell that replicate in vitro in a nutritive media. The in vitro growth rate of a varicella zoster virus can be measured as described herein.
As used herein, the term xe2x80x9cvaccine compositionxe2x80x9d refers to a pharmaceutical composition containing an antigen, where the composition can be used to prevent or treat a disease or condition in a subject. xe2x80x9cVaccine compositionxe2x80x9d thus encompasses both subunit vaccines, as described below, as well as compositions containing whole killed, attenuated or inactivated virus. xe2x80x9cSubunit vaccine compositionxe2x80x9d refers to a composition containing at least one immunogenic polypeptide, but not all antigens, derived from a varicella zoster virus. Such a subunit vaccine composition is substantially free of intact virus particles. Thus, a xe2x80x9csubunit vaccine compositionxe2x80x9d is prepared from an isolated, preferably purified, immunogenic polypeptide from the virus. A subunit vaccine composition can comprise the subunit antigen or antigens of interest isolated from other antigens or polypeptides from the pathogen.
As used herein, an xe2x80x9cattenuated varicella zoster virusxe2x80x9d refers to a varicella zoster virus that is less virulent in humans and preferably, when introduced to a human in the appropriate manner, causes a protective immunological response such that resistance to infection will be enhanced and/or the clinical severity of the disease reduced.
As used herein, a xe2x80x9csingle nucleotide polymorphismxe2x80x9d and a xe2x80x9csingle amino acid polymorphismxe2x80x9d refers to a specific type of polymorphism in a polynucleotide and a polypeptide, respectively, and are described in greater detail herein.
As used herein, the term xe2x80x9cre cognition sequencexe2x80x9d refers to the site on a polynucleotide to which a restriction endonuclease binds prior to cleaving the polynucleotide.
Unless other wise specified, xe2x80x9ca,xe2x80x9d xe2x80x9can,xe2x80x9d xe2x80x9cthe,xe2x80x9d and xe2x80x9cat least onexe2x80x9d are used interchangeably and mean one or more than one.